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Abstract:

Methods and kits are provided for assessing radiation injury and exposure
in a subject. The methods comprise measuring the levels of at least two
(2) protein biomarkers from different biological pathways and correlating
the levels with an assessment of radiation injury and exposure.
Additional use of peripheral blood cell counts and serum enzyme
biomarkers, evaluated in the early time frame after a suspected radiation
exposure, and use of integrated multiple parameter triage tools to
enhance radiation exposure discrimination and assessment are also
provided. The information obtained from such methods can be used by a
clinician to accurately assess the extent of radiation injury/exposure in
the subject, and thus will provide a valuable tool for determining
treatment protocols on a subject by subject basis.

Claims:

1. A method for assessing radiation injury and exposure in a subject
comprising measuring the levels of at least two protein biomarkers in a
test sample from the subject and correlating the levels of the at least
two biomarkers with an assessment of radiation injury and exposure.

2-3. (canceled)

4. The method of claim 1, wherein the biomarkers are from different
biological pathways.

14. The method of claim 1, wherein the test sample is selected from the
group consisting of saliva, blood, plasma, serum, skin, and urine.

15. The method of claim 1, wherein the levels of the at least two protein
biomarkers are measured with an assay selected from the group consisting
of ELISA, microsphere-based immunoassay, lateral flow test strips,
Western blots, and antibody-based dot blots.

16. The method of claim 1, wherein the levels of the at least two protein
biomarkers are measured at least 24 hours after suspected radiation
exposure.

17. The method of claim 1, wherein the levels of the at least two protein
biomarkers are measured at least 48 hours after suspected radiation
exposure.

18. The method of claim 1, further comprising measuring at least one
hematological parameter in the test sample and correlating the
hematological parameter and the levels of the at least two biomarkers
with an assessment of radiation injury and exposure.

21. The method of claim 19, wherein the peripheral blood counts comprise
one or more of neutrophil levels, lymphocyte levels, platelet levels,
and/or the ratio of neutrophil levels to lymphocyte levels.

22. (canceled)

23. The method of claim 1, further comprising assessing the dose of
radiation that the subject was exposed to and correlating the dose of
radiation and the levels of the at least two biomarkers with an
assessment of radiation injury and exposure.

24.-26. (canceled)

27. A kit for assessing radiation injury and exposure comprising
antibodies specific for at least two protein biomarkers and reagents for
conducting an immunoassay.

28.-35. (canceled)

36. The kit of claim 27, further comprising a device for measuring at
least one hematological parameter.

37.-40. (canceled)

Description:

FIELD OF THE INVENTION

[0002] This application relates generally to the rapid assessment of
radiation injury and radiation exposure and, in particular, to the use of
a panel of two or more biomarkers for assessing radiation injury and
radiation exposure.

BACKGROUND OF THE INVENTION

[0003] Current events throughout the world underscore the growing threat
of different forms of terrorism, including radiological or nuclear
attack. In the event of an attack, radiation exposures will be
heterogeneous in terms of both dose and quality, depending on the type of
device used and each victim's location relative to the radiation source.
Acute effects of high-dose radiation include changes in peripheral blood
cell numbers, immune suppression, mucosal damage (gastrointestinal and
oral), and potential injury to other sites such as the bone marrow, lung,
kidney and central nervous system (CNS). Long-term effects, as a result
of both high- and low-dose radiation, include dysfunction or fibrosis in
a wide range of organs and tissues and cancer. Early triage of suspected
radiation over-exposed individuals is needed to determine individuals
requiring immediate medical treatment.

[0004] One of the major tasks of first responders and medical personnel is
to determine the internal and external radiation doses received by
victims. This critical information provides diagnostic information to the
treating physicians and provides exposure assessments for individuals at
the site of the incident (such as first responders and medical staff).
For example, hematological and blood chemistry bioindicators have been
used in radiation exposure assessment (Blakely, 2005). Blood lymphocyte
counts decline after radiation exposure in a dose dependent manner. In
parallel, neutrophil (granulocyte) cell counts demonstrate an early rise
followed by a steep decline following medically significant radiation
exposure doses. Blakely and colleagues have developed radiation casualty
software applications, including: Biodosimetry Assessment Tool (BAT)
(Sine, 2001; Salter, 2004) and First-Responder Radiological Assessment
Triage (FRAT), using MS Windows or Palm-based operating systems to
support medical recording and triage. In the FRAT application, a multiple
parameter triage feature permits an integrated and weighted assessment of
these various biological exposure indicators.

[0005] The current methods used for estimating the radiation dose include
time to emesis, lymphocyte depletion kinetics, cytogenetic changes, and
location-based or physical dosimeter-based dose estimates. The currently
available methodologies, however, are lacking the necessary quantitative
indices to rapidly identify exposed individuals, as well as those who
could benefit from immediate medical treatment.

[0006] Ionizing radiation elicits a number of detectable changes at the
molecular, cellular and physiological level in exposed organisms. These
biological parameters have been called biomarkers. Biomarkers of
radiation exposure are biological parameters for which a dose-response
relationship can be established and can be broadly referred to as
biodosimeters. One such biodosimeter is the effect of ionizing radiation
on expression patterns of proteins, as well as modifications in proteins.

[0007] The human genome has some 30,000 to 50,000 genes that represent the
template for many more proteins, generally with proteomic patterns
specific to cell types and tissues. Biological monitoring of molecular
biomarkers can provide valuable radiation exposure assessment.

[0008] Radiation-responsive protein targets, typically measured in
peripheral blood but in certain cases other body fluids (urine, saliva,
etc.) are measured using immunoassays, including the conventional
sandwich or variations of the enzyme-linked immunosorbent assay (ELISA),
microsphere-based immunoassay (Luminex), lateral flow test strips,
protein arrays, etc. However, as noted above, the measurement of any one
radiation-responsive protein target alone does not provide the necessary
quantitative indices to identify individuals exposed to radiation.

[0009] Hoffman and colleagues reported radiation-induced increases of
serum salivary amylase in 41 patients, following either whole-body
irradiation or irradiation of the head and neck region (Hoffmann, 1990).
Becciolini and colleagues advocate the use of biochemical (e.g., serum
salivary amylase and tissue polypeptide antigen) dosimetry for prolonged
spaceflights (Becciolini, 2001). Bertho and colleagues irradiated
non-human primates at doses ranging from 2 to 8 Gy, using whole-body or
partial-body irradiation to assess a candidate plasma protein biomarker
(Flt3-ligand) as an indicator of bone marrow damage for the management of
accidental radiation-induced aplasia (Bertho, 2001). C-reactive protein
(CRP) and other serum biomarkers, derived primarily from the liver, of
acute phase reaction or inflammation have been proposed as radiation
biodosimeters (Mal'tsev, 1978; Koc, 2003).

[0010] There remains a need in the art for a rapid means of assessing
radiation injury and exposure in a patient, so that the most effective
treatment can be provided to the subject. Although the prior art methods
of measuring a single biomarker provide some information regarding a
subject's exposure to radiation, the information provided is not
sufficient to make an adequate diagnosis of the level of the subject's
exposure. Furthermore, the information provided is not sufficient to help
a clinician develop the best possible means of treatment for each subject
individually and depending on their level of exposure.

SUMMARY OF THE INVENTION

[0011] The invention provides methods for assessing radiation injury and
exposure in a subject comprising measuring the levels of at least two
protein biomarkers in a test sample from the subject and correlating the
levels of the at least two biomarkers with an assessment of radiation
injury and exposure. In some embodiments, at least three protein
biomarkers are measured, and in other embodiments, at least four protein
biomarkers are measured.

[0012] The biomarkers can be from different biological pathways. For
example, protein biomarkers such as salivary amylase, diamine oxidase,
GADD45α, p21.sup.Cif1/Waf1, p53, C-reactive protein, IL-6,
Flt-3-ligand, TNF-α, alkaline phosphatase, Raf, Bax, Bcl-2, lipase,
and citrulline can be measured in the methods of the invention.

[0013] In some embodiments of the invention, the at least two protein
biomarkers can be C-reactive protein and salivary amylase, C-reactive
protein and IL-6, or salivary amylase and IL-6. In other embodiments, the
at least three protein biomarkers can be salivary amylase, C-reactive
protein, and IL-6. In further embodiments, the at least four protein
biomarkers can be p53, salivary amylase, C-reactive protein, and IL-6 or
p21.sup.Cif1/Waf1, salivary amylase, C-reactive protein, and IL-6.

[0014] The methods of the invention can be used on test samples from
mammal subjects, such as human subjects. The test samples can be samples
such as saliva, blood, plasma, serum, skin, and urine from the subject.

[0015] The levels of protein biomarkers can be measured with assays such
as ELISA, microsphere-based immunoassays, lateral flow test strips,
Western blots, and antibody-based dot blots. In some embodiments, the
levels of protein biomarkers are measured at least 24 hours after
suspected radiation exposure, and in some embodiments, the levels of
protein biomarkers are measured at least 48 hours after suspected
radiation exposure.

[0016] Another aspect of the invention provides methods for assessing
radiation injury and exposure further comprising measuring at least one
hematological parameter in a test sample. In these methods, the
hematological parameter and the levels of at least two biomarkers are
correlated with an assessment of radiation injury and exposure. The
hematological parameter can be, for example, peripheral cell counts such
as one or more of neutrophil levels, lymphocyte levels, platelet levels,
and/or the ratio of neutrophil levels to lymphocyte levels. The
hematological parameter can also be, for example, the level of Acute
Phase Reaction biomarkers, such as C-reactive protein.

[0017] Another aspect of the invention provides methods for assessing
radiation injury and exposure further comprising assessing physiological
signs and symptoms exhibited by the subject. In these methods, the
physiological signs and symptoms and the levels of at least two
biomarkers are correlated with an assessment of radiation injury and
exposure.

[0018] Yet another aspect of the invention provides methods for assessing
the dose of radiation that the subject was exposed to. In these methods,
the dose of radiation and the levels of at least two biomarkers are
correlated with an assessment of radiation injury and exposure. The dose
of radiation can be assessed by physical dosimetry-based estimates,
including location-based estimates.

[0019] Yet a further aspect of the invention provides methods for
assessing radiation injury and exposure in a subject comprising one or
more of the steps of: measuring the levels of at least two protein
biomarkers in a test sample from the subject, measuring at least one
hematological parameter in the test sample, assessing physiological signs
and symptoms exhibited by the subject, and assessing the dose of
radiation that the subject was exposed to; and correlating one or more
of: the levels of the at least two biomarkers, the at least one
hematological parameter, the physiological signs and symptoms, and/or the
dose of radiation that the subject was exposed to with an assessment of
radiation injury and exposure.

[0020] Also provided by the invention are kits for assessing radiation
injury and exposure comprising antibodies specific for at least two
protein biomarkers and reagents for conducting an immunoassay. In some
embodiments, the kits further comprise antibodies specific for a third
protein biomarker, and in other embodiments, the kits further comprise
antibodies specific for a fourth protein biomarker.

[0021] The kits can comprise reagents for conducting immunoassays, such as
ELISA or microsphere-based immunoassays. The kits can further comprise
reagents for lateral flow test strips. At least a portion of the
antibodies for the protein biomarkers can be embedded in a lateral flow
test strip.

[0022] In some embodiments, the kits can further comprise a device for
measuring at least one hematological parameter, such as peripheral blood
counts and/or Acute Phase Reaction biomarker levels. The device can be,
for example, a finger-stick device. Peripheral blood counts can be one or
more of neutrophil levels, lymphocyte levels, platelet levels, and/or the
ratio of neutrophil levels to lymphocyte levels, and the Acute Phase
Reaction biomarker can be C-reactive protein.

[0023] The various features of novelty which characterize the invention
are pointed out with particularity in the claims annexed to and forming a
part of this disclosure. For a better understanding of the invention, its
advantages, and specific objects attained by its uses, reference is made
to the accompanying drawings and descriptive matter in which preferred
embodiments of the invention are illustrated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows radiation-responsive changes in the GADD45α
levels in human blood cells at 24 hours after exposure to 60Co gamma
rays. Each symbol represents the mean of three independent experiments
and error bars the standard error of means.

[0025] FIG. 2 shows radiation-responsive changes in the p21 WAF1 levels in
human blood cells from two different donors at 24 hours after exposure to
60Co gamma rays. Each symbol represents the mean of three
independent experiments and error bars the standard error of means.

[0026] FIG. 3 shows radiation-responsive changes in the p53 levels in
human blood cells at 24 hours after exposure to 60Co gamma rays.
Each symbol represents the mean of three independent experiments and
error bars the standard error of means.

[0027] FIG. 4 shows radiation-responsive changes in the IL-6 levels in
human blood cells at 24 hours after exposure to 60Co gamma rays.
Each symbol represents the mean of three independent experiments and
error bars the standard error of means.

[0035] FIG. 12 shows discriminant analysis data for 4 biomarkers: p21
WAF1, serum amylase, C-reactive protein, and IL-6 from monkey samples: A)
before and 1 day after, and B) before and 2 days after 6 Gy whole body
x-ray irradiation. The results from (A) show 100% discrimination power
and the results from (B) show 95% discrimination power.

[0038] FIG. 15 shows discriminant analysis data for 4 biomarkers: p53,
serum amylase, C-reactive protein, and IL-6 from monkey samples: A)
before and 1 day after, and B) before and 2 days after 6.5 Gy whole body
gamma irradiation. The results from both (A) and (B) show 100%
discrimination power.

[0042] FIG. 19 shows discriminant analysis data for C-reactive protein,
amylase activity, neutrophils, lymphocytes, and the ratio of neutrophils
to lymphocytes from monkey samples: A) before and 1 day after, and B)
before and 2 days after 6.6 Gy whole body gamma irradiation.

[0044] For the purposes of promoting an understanding of the principles of
the invention, reference will now be made to specific embodiment and
specific language will be used to describe the same. It will nevertheless
be understood that no limitation of the scope of the invention is thereby
intended, such alteration and further modifications of the invention, and
such further applications of the principles of the invention as
illustrated herein, being contemplated as would normally occur to one
skilled in the art to which the invention relates.

[0045] All terms as used herein are defined according to the ordinary
meanings they have acquired in the art. Such definitions can be found in
any technical dictionary or reference known to the skilled artisan, such
as the McGraw-Hill Dictionary of Scientific and Technical Terms
(McGraw-Hill, Inc.), Molecular Cloning: A Laboratory Manual (Cold Springs
Harbor, N.Y.), Remington's Pharmaceutical Sciences (Mack Publishing,
Pa.), and Stedman's Medical Dictionary (Williams and Wilkins, MD). These
references, along with those references and patents cited herein are
hereby incorporated by reference in their entirety.

[0047] The methods of the invention provide for assessment of radiation
injury and exposure in a subject comprising measuring the levels of at
least two (2) biomarkers and correlating the levels of the at least two
biomarkers with an assessment of radiation injury and exposure. The
methods of the invention can also comprise measuring the levels of at
least three (3) biomarkers or at least four (4) biomarkers. The
measurement of two or more biomarkers provides more detailed information
regarding the extent of a subject's radiation injury/exposure than the
prior art methods, which measured only one biomarker.

[0048] While there are many biomarkers that could be evaluated, the
operational goal is to identify the minimum number of biomarkers that
provide sufficient statistical robustness to effectively triage suspect
radiation casualties for either follow-on more demanding diagnostic
evaluation and/or early treatment decisions. Here data is provided
showing that four protein biomarkers were 100% effective to discriminate
1-day irradiated from non-irradiated samples. Data is also provided
showing that four protein biomarkers were 95% effective to discriminate
2-day irradiated from non-irradiated samples. In these cases, additional
biomarkers do not provide added value.

[0049] Radiation exposure affects different pathways over periods of time.
The dose and time dependency for the levels of blood protein biomarkers
in humans is limited but suggest that some biomarkers are more sensitive
to radiation exposure than others. Use of a non-human primate model
affords an alternative in vivo radiation model to assess the levels of
radiation-responsive blood protein biomarkers. Elevation of individual
blood protein biomarkers are not uncommon and could be attributed to one
of many non-radiation syndromes. A pattern showing elevation of one
biomarker and not the other three tested from different radiation
responsive pathways, would be considered to be inconsistent with
significant whole-body radiation overexposures. Use of multiple protein
biomarkers from different pathways present the basis for improved
diagnostics of radiation injury and exposure assessment. This more
detailed information provides the clinician more information regarding
the extent of a subject's radiation exposure, and thus allows the
clinician to make more appropriate treatment protocols on a patient by
patient basis.

[0050] Biomarkers that can be measured in connection with the methods of
the invention include, but are not limited to, salivary amylase, diamine
oxidase, GADD45α, p21.sup.Cif1/Waf1, p53, C-reactive protein, IL-6,
Flt-3-ligand, TNF-α, alkaline phosphatase, Raf, Bax, Bcl-2, lipase,
and citrulline. These radiation-responsive proteins are derived from
tissues including: parotid gland, hematological tissues, intestine,
liver, pancreas, nervous system, etc., as well as varied biological
pathways including: DNA damage and repair, cell-cycle progression,
cytokine, oncogenes, proteins along these biological pathways, or
radiosensitive tissues can be used as biomarkers. Table 1 presents a
selected list of radiation-responsive protein biomarkers and their
respective tissue or cell location.

[0052] P53 is a tumor suppressor protein that plays a major role in
cellular response to DNA damage and other genomic aberrations. Activation
of p53 can lead to either cell cycle arrest and DNA repair or apoptosis.
After exposure to ionizing radiation p53 proteins accumulate in the
nucleus and transactivate specific target genes. The p53 tumor suppressor
is required for one such G1 checkpoint and functions to upregulate
expression of GADD45α and p21.

p21 WAF1/CIP1

[0053] The cyclin-dependent kinase inhibitor p21 WAF1/CIP1, also known as
wild-type p53-activated factor (WAF-1) and Cdk-interacting protein
(CIP1), plays a critical role in cell differentiation. It also has been
shown to confer resistance to apoptosis. P21 is induced by both
p53-dependent and -independent mechanisms following stress. Induction of
p21 may cause cell-cycle arrest. This expression usually is in response
to DNA damage caused by ionizing radiation and cytotoxic agents. The
expression of p21 interrupts the cell cycle and prevents the replication
of cells containing genomic errors (Gartel, 2002; Bellido, 1998).

GADD45α

[0054] GADD45α (growth arrest and DNA damage inducible gene 45) is a
multifunctional protein that is regulated by p53 and that may play a role
in DNA replication and repair. There is evidence for its involvement in
growth control, maintenance of genomic stability, cell cycle control, and
apoptosis. GADD45α has been shown to interact with a number of
proteins playing central roles in these cellular processes: such as
proliferating cell nuclear antigen and p21 WAF1/CIP1.

Salivary α-Amylase

[0055] Apoptosis, or programmed cell death, can also occur after exposure
to ionizing radiation. Some human cells are particularly sensitive to low
levels of radiation. Once exposed to radiation, these cells exhibit
activation of a signaling cascade that leads to DNA fragmentation and
rapid cell death. Human cells that undergo radiation-induced apoptosis
include lymphocytes and acinar cells of the salivary glands. Salivary
α-amylase is a digestive enzyme secreted by salivary glands. The
salivary glands in humans appear particularly radiosensitive and the
effects of ionizing radiation can be evaluated by means of the
determination of serum amylase. Elevations in serum amylase levels may
occur within hours to days (Becciolini, 1980, 1984, 1987; Tomassi, 1979;
Chen, 1973; Van Den Brenk, 1969). Patients submitted to external
radiotherapy for tumors localized in the head and neck region show early
and late effects on salivary glands. The modification of amylase activity
appears as a progressive, statistically significant increase within two
days. Levels of 200-300% of baseline values are reached, followed by a
rapid return to pre-irradiation levels (Becciolini, 1980).

Cytokines (IL-6, Flt-3-ligand)

[0056] Secreted by macrophages, the pro-inflammatory cytokine Interleukin
6 (IL-6) induces acute-phase reaction and plays an important role in
immunity. Interleukin 6 (IL-6) is involved in p53-independent activation
of p21 via different transcription factors (Gartel, 2002; Bellido, 1998).
IL-6 is a chief stimulator of the production of most acute-phase
proteins. IL-6 was shown to be an essential contributor for natural
resistance to lethal irradiation. Expression of cytokines such as IL-6,
IL-1, and TNFα as a result of inflammation through vascular and
hematological damage is induced by radiation injury. The cytokines such
as IL-6, TNF, and IL-1 may be useful to protect hematopoietic cells from
radiation, while TNF may enhance the killing of tumor cells. Bertho and
colleagues reported results for irradiated non-human primates at doses
ranging from 2 to 8 Gy, using whole-body or partial-body irradiation to
assess a candidate plasma protein biomarker Flt3-ligand as an indicator
of bone marrow damage for the management of accidental radiation-induced
aplasia (Bertho, 2001).

Acute-Phase Proteins CRP and SAA

[0057] The cytokines that are produced during and participate in
inflammatory processes are the chief stimulators of the production of
acute-phase proteins. The most notable acute-phase proteins synthesized
by liver are C-reactive protein (CRP) and Serum Amyloid A (SAA).

[0058] CRP is a sensitive biomarker of disease with a broad clinical
utility for cardiovascular injury monitoring and differential diagnosis.
CRP plays an essential role in injuries caused by radiation (Wood, 1960;
Tukachinski, 1961; Mal'tsev, 1978; Koc, 2003). Mal'tsev and colleagues
reported data for CRP content in blood of 147 patients damaged at
Chernobyl NPP accident as a result of radiation exposure. They determined
that indexes of CRP content in a peripheral blood during primary reaction
on the irradiation (1-2 days after) and in the latent period of radiation
disease (3-9 days after) can provide the information for the prognosis of
probable gravity (level) of radiation injury (Mal'tsev, 2006).

[0059] During acute events, the rise in the level of SAA in blood exhibits
the most intense and rapid increase among all acute-phase proteins.
Cytokines such as IL-1, IL-6, and TNF are considered mediators of SAA
protein synthesis. Induction of serum amyloid A inflammatory response
genes in bone marrow cell of mice exposed to total-body irradiation was
reported by (Goltry, 1998). Significant elevation of serum amyloid A has
been found in irradiated mice and cancer patients treated with a
synthetic compound AS101 with radioprotective and chemoprotective effects
(Kalechman, 1995).

Ferritin

[0060] Hematological biomarkers of exposure to ionizing radiation are well
characterized and used in medical management of radiological casualties
(Dainiak 2002). The combination of intestinal and hemopoietic syndromes
results in dehydration, anemia, and infection, leading eventually to
irreversible shock and death. Change in iron metabolism parameters
(increased level of serum ferritin, appearance of an iron pool
specifically unrelated to transferrin, reduced ceruloplasmin level, etc.)
as affected by ionizing radiation were found changed in subjects who
participated in liquidation of Chernobyl power plant accident
aftereffects (Levina, 1993). The effect of iron release from ferritin
using 137Cs gamma radiation was investigated by Reif et al. (Reif,
1988). Observations over the last few years have placed the regulation of
ferritin within the broad context of cell injury and stress as well as
altered growth regulation (Torti, 2002).

[0061] Ferritin is a ubiquitous and highly conserved iron-binding protein.
Ferritin also has enzymatic properties, converting Fe(II) to Fe(III) as
iron is internalized and sequestered in the ferritin mineral core.
Elemental iron is required for a variety of normal cellular functions. It
is vital for proper growth and development. Iron is involved in the
formation of oxidants capable of damaging membranes, protein, and DNA.
Ferritin is a major protein involved in iron sequestration and
detoxification. Increasingly, perturbations in cellular iron and ferritin
are emerging as an important element in the pathogenesis of disease.
These changes in ferritin are important not only in the classic diseases
of iron acquisition, transport, and storage, such as primary
hemochromatosis, but also in diseases characterized by inflammation,
infection, injury, and repair.

[0062] The critical role of ferritin in cellular and organism iron
homeostasis is intimately linked to its primary and best-studied
function--iron sequestration. Iron in heme is necessary for the
transport, binding, and release of oxygen; the ready availability of iron
for incorporation to heme is essential to organism survival. Iron is also
essential for the function of enzymes that participate in numerous
critical cellular processes, including the cell cycle, the reductive
conversion of ribonucleotides to deoxyribonucleotides, electron
transport, and others.

[0063] One of the major functions of ferritin is to limit Fe(II) available
to participate in the generation of oxygen-free-radicals (ROS). Oxidant
stress is an ever-present threat to organism survival, both from
exogenous and endogenous cellular sources; it is therefore not surprising
that oxidant stress activates multiple pathways of ferritin regulation.
Small quantities of ferritin are present in human serum, and are elevated
in conditions of iron overload and inflammation (Lipschitz, 1974; Koziol,
2001; Torti, 1994).

[0064] Secretion of ferritin is stimulated by cytokines. Cytokines play a
pivotal role in the cellular response to infection, and ferritin plays a
prominent role in the cytokine response. TNFα, and interleukin
1α (IL-1α), another proinflammatory cytokine,
transcriptionally induce the H chain of ferritin, suggesting that
pathways related to inflammation and stress can impact on ferritin
regulation (Torti, 1988; Wei, 1990).

[0065] Cytokines also have transcriptional effects on ferritin in
different cell types. Ferritin induction in macrophages may be
particularly important, given their central role in iron homeostasis as
scavengers of old and damaged red blood cells, a critical and
quantitatively important element in whole-body iron turnover. Cytokines
also regulate ferritin posttranscriptionally: induction of ferritin
synthesis was observed with a number of cytokines: IL-1β, IL-6,
TNFα.

[0066] In some cell types, ferritin has been observed to increase in
growth arrest (Larsson, 1998). Growth suppression associated with
overexpression of ferritin H has also been reported (Cozzi, 2000).
Upregulation of ferritin was also associated with induction of
differentiation and growth arrest in hematopoetic systems (Thweatt,
1992).

Citrulline

[0067] Citrulline is an α-amino acid that is produced as an
intermediate in the conversion of ornithine to arginine during urea
formation in the liver. It is also produced from arginine as a by-product
of the reaction catalyzed by nitric oxide synthase. Plasma citrulline has
been found to be an effective protein marker for quantitation and
monitoring of epithelial radiation-induced small bowel damage (Lutgens,
2003; Lutgens, 2004).

[0068] The biological samples which can be obtained from a subject and
used for the methods of the invention include, but are not limited to,
blood, plasma, serum, saliva, skin, urine, hair follicles, and other
accessible tissues. The choice of biological sample used can depend on
the biomarker that will be measured. For example, tissue from the parotid
gland is used to measure salivary amylase activity, however, radiation
issue is known to cause proteins from the parotid gland to be released in
peripheral blood and then detected as an indicator of radiation
overexposure (Becciolini, 2001). Skin tissue can be used to measure
cytokine levels (IL-6, Flt-3-ligand, TNF-α, etc.). Blood can also
be used to measure cytokine levels, as well as alkaline phosphatase,
GADD45α and p21.sup.Cif1/Waf1 levels. Liver tissue can be used to
measure C-reactive protein (CRP) but as in the case of amylase above,
following radiation exposure blood plasma levels of CRP are significantly
elevated at one and two days after 6.5-Gy radiation exposure in non-human
primate (Macacq mulatta) whole-body radiation models.

[0069] The use of hematological and serum enzyme activity biomarkers,
evaluated in the early time frame after a suspected radiation exposure,
in combination with the use of integrated multiple parameter triage tools
can enhance radiation exposure discrimination and assessment. Examples of
hematological parameters that can be evaluated include peripheral cell
counts and/or Acute Phase Reaction biomarker levels. Peripheral cell
counts include, for example, peripheral blood counts comprise one or more
of neutrophil levels, lymphocyte levels, platelet levels, and/or the
ratio of neutrophil levels to lymphocyte levels. Acute Phase Reaction
biomarkers include, for example, C-reactive protein.

[0070] Accordingly, also provided by the invention are methods of
assessing radiation injury and exposure in a subject comprising measuring
the levels of at least two protein biomarkers and measuring at least one
hematological parameter in a test sample and correlating the levels of
the at least two protein biomarkers and the at least one hematological
parameter with an assessment of radiation injury and exposure.

[0071] The invention further provides methods of assessing radiation
injury and exposure in a subject comprising assessment of other
diagnostic information indicative of radiation exposure. For example, in
addition to hematological parameters, assessment of physiological signs
and symptoms exhibited by the subject and an estimate of the dose of
radiation that the subject was exposed to can be integrated with results
of levels for multiple protein targets to improve the assessment of
radiation injury and exposure. Physiological signs and symptoms that may
be indicative of radiation exposure include signs and symptoms relating
to the subject's neurovascular system (e.g. nausea, vomiting, anorexia,
fatigue syndrome, fever, headache, hypotension, neurological deficits,
cognitive deficits), hematopoietic system (e.g. lymphocyte changes,
granulocyte changes, thrombocyte changes, blood loss, infection),
cutaneous system (e.g. erythema, sensation/itching, swelling/edema,
blistering, desquamation, ulcer/necrosis, hair loss, onycholysis), and/or
gastrointestinal system (e.g. diarrhea, abdominal cramps/pain). An
estimate of the dose of radiation that the subject was exposed to can be
obtained, for example, by physical dosimetry based on personnel dosimeter
or location-based estimates. Any method of assessing radiation injury and
exposure comprising measuring the levels of at least two protein
biomarkers combined with assessing one or more other diagnostic
parameters indicative of radiation exposure is contemplated by the
invention.

Assays and Kits for Measuring Protein Biomarkers

[0072] Methods for measuring the amount of biomarker present in a sample
include, but are not limited to, ELISA, microsphere-based immunoassay,
lateral flow test strips, antibody based dot blots or Westerns.
Antibodies which can be used in any of these immunoassays include, but
are not limited to, monoclonal or polyclonal antibodies to salivary
amylase, IL-6, Flt-3 ligand, TNF-α, alkaline phosphatase,
GADD45α, p21.sup.Cif1/Waf1, C-reactive protein, Raf, Bax, Bcl-2,
P53, lipase, etc. Chromatography (i.e., HPLC, GC, etc.) based separation
and subsequent detection during various sensors (i.e., UV, fluorescence,
etc.) as well as 2D-gel/matrix assisted laser desorption/ionization
represents examples of alternative methods to measure protein levels for
diagnostic radiation exposure assessment that does not require use of
antibodies.

[0073] Also provided are kits for assessing radiation injury and exposure
in a patient. Said kits comprise antibodies specific for a first
biomarker; antibodies specific for a second biomarker; and reagents for
conducting an immunoassay. The kits can further comprise antibodies for a
third biomarker, and preferably will also comprise antibodies specific
for a fourth biomarker. There can be more than one set of reagents
present in the kit. For example, if the kit is intended for use with just
ELISA, than only reagents for ELISA will be present. If the kit is
intended for use with both ELISA and a microsphere-based immunoassay,
then reagents for both ELISA and microsphere-based immunoassay will be
present. If the kit is intended for use with ELISA, a microsphere-based
immunoassay, and with lateral flow test strips, then reagents for all
three immunoassays will be present, etc.

[0074] If lateral flow test strips are to be used to conduct the
immunoassay, then the antibodies within the kit will be embedded in the
lateral flow test strips. Also, the kits can be intended for use with a
combination of immunoassays. So, for example, the antibodies for the
first biomarker may be present in the kit as free antibodies, but a
portion of them may also be embedded in a lateral flow test strip. The
same can be said for the antibodies for the second biomarker, the third
biomarker, and the fourth biomarker, etc.

[0075] The kits may include devices for measuring hematological
parameters, such as peripheral blood counts and Acute Phase Reaction
biomarkers. Such devices can be modifications of commercially available
devices for assessing blood cell counts and APR biomarkers, such as the
Quikread CRP finger-prick device (Orion Diagnostica, Finland) which
measures the level of C-reactive protein.

[0076] Reference will now be made to specific examples illustrating the
constructs and methods above. It is to be understood that the examples
are provided to illustrate preferred embodiments and that no limitation
to the scope of the invention is intended thereby.

EXAMPLES

[0077] Experimental protocols used to perform the studies that generated
the data disclosed include a) radiation model systems and b) biomarker
assays.

[0078] Radiation models employed include use of: a) human ex vivo
radiation models, b) murine (Mus musculus) in vivo radiation models, and
non-human primate (NHP) (Macaca mulatta) in vivo radiation models. In the
human ex vivo radiation model, peripheral blood is drawn typically from
healthy human donors, exposed ex vivo to ionizing radiation, and then
incubated at 37° C. for various intervals. In the murine and NHP
in vivo radiation models the animals are typically exposed to ionizing
radiation and then blood is drawn at various times after exposure.

[0080] Radiation responsive protein biomarkers were measured using ELISA
and as well as a novel and high-throughput microsphere-based
multi-analyte Luminex assay system. An in vitro model system of human
peripheral blood lymphocytes showed radiation-responsive changes in the
expression of GADD45α, p21 WAF1/CIP1, p53, and IL-6 with a
progressive time- and dose-dependent increase (FIGS. 1-5). Protein levels
were determined using a calibration curve with a reference standard.
Symbols represent the mean of three independent experiments and error
bars the standard error of means.

[0081] Induction of these proteins by low-dose radiation has a different
dependence on the time after irradiation than induction by high doses.
Dose dependent increases in presented data show the potential utility of
these protein biomarkers to detect radiation exposure. A retrospective
correlation analysis led to the finding of strong correlations between
different combinations of presented candidate radiation-responsive blood
protein biomarkers (FIG. 6 and FIG. 7).

[0084] Data analyzed with use of multivariate discriminant analysis
established very successful separation of non-human primate groups: 100%
discrimination power for animals with correct classification for
separation between groups before and 1 day after irradiation and 95%
discrimination power for animals with correct classification for
separation between groups before and 2 days after irradiation. (FIG. 12).
Clear separation of animals before and after irradiation can be seen. The
plot in FIG. 13 presents a result of classification and discrimination
analyses for different combinations of biomarkers for 1 and 2 days after
6 Gy whole body irradiation. A progressive increase in the number of
biomarkers from one to 4 improved the ability to discriminate control
from exposed samples.

[0085] Multivariate discriminant analysis was performed using the
Statistical Analysis Software (SAS) package to analyze the data for
different combinations and number of measured biomarkers to separate
monkey cohorts for different time points before and after 6-Gy whole-body
irradiation. The DISCRIM procedure in SAS/STAT® calculates the
posterior probability of each individual animal belonging to each of
three subgroups (cohorts) and assigns the subject to a corresponding
subgroup according to the higher probability. In addition, the DISCRIM
procedure summarizes the squared distance between subgroups, univariate
and multivariate statistics, canonical coefficients to derive canonical
variables (a dimension-reduction technique), the list of misclassified
observations, classification error-rate, the result of classification for
each subject, and total frequency of separation. The purpose of the
canonical score is to make separation between the classes as large as
possible. Canonical scores represent the observation in the
multidimensional space and can be positive or negative. Canonical scores
have been used for 2D b-plots to aid the visual interpretation of group
differences.

[0088] Domestic-born, male rhesus monkeys, Macaca mulatta, 4.8±0.7 kg,
were housed in individual stainless steel cages in conventional holding
rooms at the University of Maryland, Veterinary Resources Department in
an animal facility accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care International (AAALAC). Research
was conducted according to the principles enunciated in the Guide for the
Care and Use of Laboratory Animals, prepared by the Institute of
Laboratory Animal Resources, National Research Council, and under an
IACUC approved protocol. Rhesus monkeys received TBI to a midline tissue
dose of 600 cGy, 250-kVp x-irradiation at 13 cGy min-1. The dose (6
Gy) was selected to allow detection of a sufficient signal above
background for whole-body exposure. Ketamine-anesthetized animals
(Ketaset® [10 mg kg-1, i.m.], Fort Dodge Laboratories; Fort
Dodge, Ind.) were placed in a plexiglass restraint chair (to which they
had been previously prehabituated), allowed to regain consciousness, and
were x-irradiated in the posterior-anterior direction, then rotated at
mid-dose to the anterior-posterior direction to complete the exposure.
Dosimetry was performed using paired 0.5-cm3 ionization chambers,
with calibration factors traceable to the National Institute of Standards
and Technology.

[0089] Blood sampling at pre-TBI and at 24 and 48 hours after were
selected for this study based on radiation accident operational
considerations. For example, generic guidelines recommend that blood for
cytogenetic analysis be collected 24 hour after a suspect radiation
exposure (IAEA, 2001). Tubes with collected peripheral blood were
centrifuged at 800 g (4° C.) for 10 min to isolate the supernatant
(plasma) and cell pellets. Blood samples were taken via aliquot and
stored at -80° C. until use.

[0090] Protein Bioassays

[0091] Samples were assayed for colorimetric detection and quantitation of
total protein via the bicinchoninic acid (BCA) method (Pierce) prior to
the immunoassay. For ELISAs, plasma samples were diluted in
phosphate-buffered saline (PBS) for equal amounts of 2.65 mg total
protein content per each well. Polysterene 96-well microtiter plates
(NUNC Brand Products, Nalge NUNC International, Rochester, N.Y.) were
used to perform immunoassays.

[0093] Three replicate measurements per each of three independent
experiments were determined for each sample. Data are presented as plasma
p21 WAF1 IgG ELISA absorbance, which represents optical density (OD)
units per equivalent total protein levels (2.65 mg) per well. The
candidate radiation-responsive blood protein biomarker salivary
α-amylase was measured in newly developed modifications of indirect
ELISA. Rabbit polyclonal anti-alpha-amylase (Cat. #A36, Biomeda, Foster
City, Calif.) was added to plasma (antigen) passively adsorbed to a solid
phase (96-well maxisorb polysterene plate). After 3 hours, incubation at
room temperature secondary-antibody-Biotin-SP-conjugated anti-rabbit IgG
(Cat. #111-065-003, Jackson ImmunoResearch Laboratories, Inc., West
Grove, Pa.) was added and incubated for 1 h at room temperature. After a
wash step, horseradish peroxidase (HRP)-conjugated streptavidin
(Streptavidin-HRP, R&D Systems, Inc., Minneapolis, Minn.) was added and
incubated at room temperature. After a final wash step, the K-Blue
substrate (Cat.#308176, Neogen Corporation, Lexington, Ky.) was added for
color development. The reaction was stopped after 30 min using a stop
solution (Cat.#301475, Neogen Corporation, Lexington, Ky.). The amount of
color that developed was measured at 650 nm in a microtiter plate using a
spectrophotometer. A human salivary α-amylase standard protein
(Cat. #A1031, Sigma-Aldrich, St. Louis, Mo.) was used to check antibody
responses.

[0094] Sandwich ELISA for monkey C-reactive protein (CRP) was performed
using a commercially available kit (Cat. #2210-4, Life Diagnostics, Inc.,
West Chester, Pa.) according to the manufacturer's instructions. The
sensitivity for assay was 0.8 ng ml-1. Three replicate measurements
were determined for each sample and standards.

[0095] A sandwich ELISA for monkey IL-6 was performed using a commercially
available kit (Cat. #CKM005, Cell Sciences, Inc., Canton, Mass.). The
sensitivity for assay was 1 pg ml-1. Three replicate measurements
were determined for each sample and standards.

[0096] The CRP and IL-6 concentrations in plasma samples were determined
via use of Table Curve 2D software.

[0097] Data Analysis

[0098] Statistical software, PC SAS was used for statistical analyses (SAS
Institute Inc., 2000; Khattree et al., 2000). Multivariate analysis of
variance (MANOVA) was used to determine if there was a significant
difference among three sampling time points in any outcome variable, by
using Wilks' Lambda statistics. If there was a significant difference
among the three days, then pairwise comparisons were used. A significance
level was set at 5% for each test. All statistical tests were two-sided.
Adjustment of multiple tests was not made. For each monkey at a given
day, average of the three observations for an outcome variable was used
for the statistical analyses. The DISCRIM procedure was used in data
analysis to separate irradiated subgroup of animals from non-irradiated.
Discriminant analysis is a multivariate statistical procedure that
mathematically defines a special discriminant function to separate study
animal groups by one classification variable (time after irradiation).

[0108] The lower right panel of FIG. 11 shows the result of measuring IL-6
expression in monkey plasma. One can see significantly increased plasma
IL-6 levels at 24 and 48 h post irradiation. Plasma IL-6 content baseline
values ranged from 1.15 (±1.09) pg ml-1 to 35.48 (±2.38) pg
ml-1 with a pooled cohort mean value of 13.34 (±4.45) pg
ml-1. In individual monkeys, radiation caused a 2.11- to 72.44-fold
increase (pooled value of 13.75-fold) in plasma CRP content at 24 h
relative to the individual baseline values. The pooled value of plasma
CRP content in the cohort (n=10) was increased by 13.75- and 27.37-fold
at 24 or 48 h, respectively, after radiation. IL-6 levels at baseline
increased at 24-h significantly and then increased at 48-h not
significantly, while the levels at 48-h significantly increased from the
baseline (p=0.0021, 0.0648, 0.0025 respectively; MANOVA p=0.0047).

[0109] Data presented show the potential utility of protein biomarkers to
detect radiation exposure. The DISCRIM procedure in SAS/STAT® was
used in data analysis by the definition a special discriminant function
to separate study animal groups by one classification variable (time
after irradiation). The discriminant function can use several
quantitative variables (biomarkers), each of them makes an independent
contribution to the overall discrimination. Taking into consideration the
effect of all quantitative variables, this discriminant function produces
the statistical decision for guessing to which subgroup of classification
variable each subject (animal) belongs to. The DISCRIM procedure in
SAS/STAT® calculates the posterior probability of each individual
animal belonging to each of three subgroups and assigns the subject to a
corresponding subgroup according to the higher probability; summarizes
the squared distance between subgroups in multidimensional (dimension is
a number of independent variables) space taking into account correlations
between variables. The DISCRIM procedure produces quantitative variables:
Wilks' Lambda that assume values in the range of 0 (perfect
discrimination) to 1 (no discrimination) and provides information about
upper limit for number of biomarkers and Partial Lambda associated with
the unique contribution of the respective variable (biomarker) to the
discriminatory power of the model. Procedure derives a list of
misclassified observations, classification error-rate, the result of
classification for each subject, and canonical scores that represent the
observation in the multidimensional space. Canonical scores have been
used for 2D-plots to aid the visual interpretation of subgroup
differences. The purpose of the canonical score is to make separation
between the classes as large as possible. Thus, when observations are
plotted with canonical scores as the coordinated, observations belonging
to the same class are grouped together. As a result of classification and
discrimination analysis, we also have a list with detailed information
for each animal: predicted and observed classification.

[0110] FIGS. 12 and 13 show the results of discriminant analysis for
different combinations and a number of biomarkers for 24 and 48 h after
6-Gy whole-body irradiation. Error bars represent the standard deviation
for discrimination power values for a given number of biomarkers. An
enhanced separation between animal groups was observed as the number of
biomarkers increased. FIG. 12 shows separation of animals before and 24 h
and 48 h after 6-Gy whole-body irradiation for four protein biomarkers:
p21 WAF1/CIP1, salivary α-amylase, CRP, and IL-6. Results
demonstrate a distinct separation of animals with 100% discrimination
power (without any overlap) between before and 24 h after irradiation.

[0112] Data analyzed with use of multivariate discriminant analysis
established very successful separation of non-human primates groups: 100%
discrimination power for animals with correct classification for
separation between groups before and after irradiation (FIG. 15). Clear
separation of animals before and after irradiation can be seen. The plot
in FIG. 16 presents a result of classification and discrimination
analyses for different combinations of biomarkers for 1 and 2 days after
whole body irradiation. A progressive increase in the number of
biomarkers from one to 4 improved the ability to discriminate control
from exposed samples.

[0113] We investigated the utility of serum amylase and hematological
blood-cell count biomarkers to provide early assessment of severe
radiation exposures in a non-human primate model (i.e., rhesus macaques;
n=8) exposed to whole-body radiation of 60Co-gamma rays (6.5 Gy, 40
cGy min-1). Serum amylase activity was significantly elevated
(12.3±3.27- and 2.6±0.058-fold of day zero samples) at 1 day and
2-days, respectively, after radiation. Lymphocyte cell counts decreased
(≦15% of day zero samples) 1 and 2 days after radiation exposure.
Neutrophil cell counts increased at day one by 1.9(±0.38)-fold
compared with levels before irradiation. The ratios of neutrophil to
lymphocyte cell counts increased by 13(±2.66)- and 4.23(±0.95)-fold
at 1 and 2 days, respectively, after irradiation. These results
demonstrate that increases in serum amylase activity along with decreases
of lymphocyte counts, increases in neutrophil cell counts, and increases
in the ratio of neutrophil to lymphocyte counts 1 day after irradiation
can provide enhanced early triage discrimination of individuals with
severe radiation exposure and injury.

[0116] Male rhesus monkeys (Macaca mulatta) (6.8 to 12.2 kg; 7 to 10 y
old; n=8), were housed in individual stainless steel cages in
conventional holding rooms at the AFRRI's Veterinary Sciences Department
in an animal facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC) International. Research
was conducted according to the principles enunciated in the Guide for the
Care and Use of Laboratory Animals, prepared by the Institute of
Laboratory Animal Resources, National Research Council.

[0117] Rhesus monkeys received myeloablative conditioning as total-body
exposure to a midline tissue dose of 6.50 Gy, 60Co-y irradiation at
40 cGy m-1. The dose (6.5 Gy) was selected as part of a medical
countermeasures study. Ketamine-anesthetized animals (Ketaset® [10 mg
kg-1, i.m.], Fort Dodge Laboratories; Fort Dodge, Ind.) were placed
in a plexiglass restraint chair (to which they had been previously
habituated), allowed to regain consciousness and were irradiated
bi-laterally. Dosimetry was performed using an alanine/electron
paramagnetic respondance system, with calibration factors traceable to
the National Institute of Standards and Technology and confirmed by an
additional check against the national standard 60Co source of the UK
National Physics Laboratory.

[0120] Complete blood cell counts and differentials were determined using
a clinical hematology analyzer (Bayer Advia 120, Bayer, Tarrytown, N.Y.).
Three replicate measurements were performed for each sample.

[0121] Serum Amylase Activity

[0122] Amylase activities from serum samples were measured using a
clinical blood chemistry analyzer (Bayer Vitros 250, Ortho-Clinical
Diagnostics, Rochester, N.Y.). Preliminary analysis determined that the
samples from irradiated animals were elevated and required 10-fold
dilutions for measurements to fall within the calibrated dose range.
Three replicate measurements were determined for each sample.

[0123] Plasma α-Amylase

[0124] The candidate radiation-responsive blood protein biomarker,
salivary α-amylase, was measured in a modification of an
enzyme-linked immunosorbent assay (ELISA). Samples are assayed for
colorimetric detection and quantitation of total protein via the
bicinchoninic acid (BCA) method (Pierce) prior to the immunoassay. For
ELISA, plasma samples were diluted in phosphate buffered saline (PBS) to
yield an equal amount (2.65 mg) of total protein content per each well
(100 μl). Polysterene 96-well microtiter plates (Nalge NUNC
International, Rochester, N.Y.) were used to perform immunoassays.

[0127] Multivariate analysis of variance (MANOVA) repeated measures
analysis was used to determine if there was a significant difference
among day 0, day 1, and day 2 in any outcome variable, by using the
Wilks' Lambda statistics. If there was a significant difference among the
three days, then we compared day 1 and day 2 results versus day 0,
respectively (Morrison, 1976). A significance level was set at 5% for
each test. All statistical tests were two-sided. A statistical software
package (Personal Computer-Statistical Analysis Software) was used for
statistical analyses.

[0128] Results

[0129] Hematology

[0130] Blood cell counts with complete white blood cell differential from
8 rhesus monkeys were obtained before and at 24- and 48-h after radiation
exposure (FIG. 17). Baseline peripheral blood-lymphocyte and neutrophil
cell numbers fell between 0.6 to 3.5×109I-1 and 0.8 to
5.4×109I-1, respectively. Lymphocytes at 24- and 48-h
after irradiation declined markedly among the cohort of 8 monkeys with a
pooled mean decline value of 85.4 (±0.32) and 83.7 (±3.41)%,
respectively. In 7 of 8 monkeys, the neutrophils at 24 h after
irradiation increased with a pooled (n=8) mean increase value of 1.9-fold
(±0.38). In 7 of 8 monkeys, neutrophils at 48 h after irradiation
either returned to or fell below (65.7±12.6%; pooled before
irradiation value) initial baseline levels.

[0131] The ratio of neutrophil to lymphocyte (N/L) cell numbers was also
evaluated (FIG. 17). Baseline N/L values ranged from 1.15 to 3.46 with a
pooled cohort value of 2.0 (±0.34). After irradiation the N/L value
increased 13- (±2.66) and 4.23-fold (±0.95) at 24-h and 48-h,
respectively, in the cohort (n=8) compared with the pre-irradiation
value.

[0132] Amylase Measurements

[0133] Serum amylase activity and plasma α-amylase protein content
were measured before as well as 24- and 48-h after irradiation of monkeys
(FIG. 18). Plasma α-amylase protein content baseline values ranged
from 0.015 to 0.085 IgG ELISA absorbance with a pooled cohort mean value
of 0.053 (±0.0106). In individual monkeys, radiation caused a 1.8- to
5.6-fold increase (pooled value of 2.73±0.44 fold) in plasma
α-amylase protein content at 24 h relative to the individual
baseline values. In 3 of 8 monkeys, α-amylase protein content was
elevated (1.45 to 2.7 fold) 48 h after radiation. The pooled value of
plasma α-amylase protein content in the cohort (n=8) was increased
by 2.39- (±0.607) and 1.42-fold (±0.38) at 24 or 48 h,
respectively, after radiation (FIG. 18) compared to the pooled cohort
value.

[0135] Blood samples from rhesus monkeys exposed to 6.5 Gy at several
sampling time-points were obtained. The levels of C-reactive protein,
amylase activity, neutrophils, lymphocytes, and the ratio of neutrophils
to lymphocytes, were measured in the samples. The data was analyzed with
use of multivariate discriminant analysis and established very successful
separation of non-human primates groups: 100% discrimination power for
animals with correct classification for separation between groups before
and 24h and 48h after irradiation (FIG. 19). Clear separation of animals
before and after irradiation can be seen.

Example 7

Dose-Response (Mus Musculus) in Vivo Radiation Model

[0136] Multiple Regression Analysis was used to develop dose-response
relationships for multiple protein inductions for radiation dose
assessment (FIG. 20). The general purpose of multiple regression is to
learn more about the relationship between several independent variables
(biomarkers) and a dependent variable (dose). Multiple regression
procedures estimate a linear equation of the form:

Y=a+b1*X1+b2*X2+ . . . +bp*Xp

where Y variable (dose assessment) can be expressed in terms of a
constant (a) and a slope (b) times the X variables (dose-response protein
expression), p is a number of protein biomarkers in the model. The
regression coefficients (or b coefficients) represent the independent
contributions of each independent variable to the prediction of the
dependent variable.

[0137] There is considerable individual variability in radiation response
that makes the diagnostic utility of individual proteins limited in
exposure dose assessment, but still feasible when analyzed according to
multiple biomarkers pathway. To our knowledge, this is the first report
of a dose-response calibration curve for multiple radiation-responsive
protein biomarkers. Use of multiple protein targets, along with classic
biodosimetric methodologies, is expected to enhance the prognostic
utility of protein-based biomarkers approach for early assessment of
severe radiation over-exposure.

[0138] While the foregoing specification teaches the principles of the
invention, with examples provided for the purpose of illustration, it
will be appreciated by one skilled in the art from reading this
disclosure that various changes in form and detail can be made without
departing from the true scope of the invention.